Project Summary Mesencephalic dopamine neurons are critical for processing reward and therefore are implicated in the rewarding and addicting properties of abused drugs. The long-term goal of the candidate is to become an independent investigator researching dopamine neuron physiology, with a focus on drugs-induced adaptations. This proposal has been designed to address key research questions while supplementing the candidate's background in molecular biology and neurophysiology with additional training in advanced light microscopy and biochemistry. This new knowledge will greatly assist in achieving the candidate's career goals. In the midbrain, dopamine neurons release dopamine from their dendrites to regulate the excitability of neighboring neurons. There is a discord in the field with structural studies indicating that midbrain dopamine transmission is paracrine in nature, while functional electrophysiology studies indicate the transmission is more synaptic. The goal of this research is to bridge the gap between inconsistent findings by employing a new mouse model with a GFP knocked-in to the endogenous D2 receptor gene. Using both confocal and 2-photon microscopy, recently published and preliminary results show that D2 receptors are clustered into puncta on dendrites and spines of midbrain dopamine neurons. Furthermore, upon exposure to a desensitizing concentration of agonist the receptors do not traffic. This contrasts with other G protein-coupled receptors (GPCRs), like the -opioid receptor, which are located uniformly on the cell membrane and internalize upon desensitization. The hypothesis is that midbrain dopamine transmission is point-to-point and that D2 receptors are anchored into synaptic protein complexes. Additionally, if D2 receptors are indeed synaptic, plasticity should be detectable at individual D2 synapses. In this proposal electrophysiology, 2-photon and confocal microscopy, and proteomics will be used to address these hypotheses. In Aim 1 (K99), caged neurotransmitters (dopamine, GABA, glutamate) will be photoactivated at synaptic locations on dopamine neurons to determine whether D2 receptors are co-localized with the receptors for synaptic excitatory or inhibitory inputs. Experiments in Aim 2 (K99) use proteomics to attempt to determine which proteins might interact with the D2 receptor, anchoring it in synaptic locations. In Aim 3 (R00), cocaine-induced plasticity at individual D2 receptor puncta will be investigated using 2-photon imaging and electrophysiological techniques. Aim 4 (R00) follows up on the identification of D2-associated proteins from the K99 phase with functional exploration of the impact of these interactions on dopamine neuron physiology. Under the mentorship and training of Dr. Williams, who has guided over 20 scientists into independent careers, with the added expertise of Dr. Zhong in fluorescence imaging and Dr. Neve in protein biology, the candidate will be in a competitive position to obtain a tenure track research position at the conclusion of the K99 phase and well prepared to succeed as an independent investigator.